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ObjectiveTo predict the potential targets and possible related signaling pathways of Salviae Miltiorrhizae Radix et Rhizoma against bladder cancer （BC） based on network pharmacology and verify the potential molecular mechanism through in vitro cell experiment. MethodActive components of Salviae Miltiorrhizae Radix et Rhizoma were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP） and BC-related targets were searched from GeneCards and Online Mendelian Inheritance in Man （OMIM）. Via Venny2.1， the potential targets of Salviae Miltiorrhizae Radix et Rhizoma against BC were screened out and the Venn diagram was plotted. Protein-protein interaction （PPI） network was constructed by STRING， followed by Gene Ontology （GO） term enrichment and Kyoto Encyclopedia of Genes and Gnomes （KEGG） pathway enrichment with DAVID. Cell Counting Kit-8 （CCK-8） assay was employed to detect the inhibitory effect of tanshinone ⅡA （Tan ⅡA）， cryptotanshinone （CPT）， and luteolin （LUT） at different concentration （0， 1， 2， 4， 8， 16， 32 μmol·L-1） on the proliferation of BC T24 and 5637 cells， propidium iodide （PI） staining to analyze the apoptosis of 5637 cells induced by Tan ⅡA， CPT， and LUT （0， 4， 8 μmol·L-1）， and Western blotting to detect the regulatory effect of Tan ⅡA （0， 4， 8， 16 μmol·L-1） on the expression of key target proteins. ResultA total of 65 active components and 39 anti-BC targets of Salviae Miltiorrhizae Radix et Rhizoma were screened out. The anti-BC targets were mainly involved in the KEGG pathways of neuron-ligand-receptor interaction， phosphatidylinositol 3-kinases （PI3K）/protein kinase B （Akt） signaling pathway， epidermal growth factor receptor （EGFR） tyrosine kinase inhibitor resistance， and hypoxia inducible factor （HIF）-1 signaling pathway. As for the CCK-8 assay， compared with the blank group， Tan ⅡA， CPT， and LUT significantly inhibited the proliferation of T24 and 5637 cells， particularly the 5637 cells. The half maximal inhibitory concentration （IC50） of Tan ⅡA on 5637 cells was significantly lower than that of CPT and LUT. Moreover， compared with the blank group， Tan ⅡA， CPT， and LUT all induced the apoptosis of 5637 cells， and the effect followed the order of Tan ⅡA>CPT>LUT （P<0.05）. Western blot showed that Tan ⅡA significantly reduced the expression of EGFR， p-PI3K， and p-Akt in 5637 cells in a concentration-dependent manner compared with the blank group （P<0.05）. ConclusionSalviae Miltiorrhizae Radix et Rhizoma exerts therapeutic effect on BC through multiple components， multiple targets， and multiple pathways. The mechanism is the likelihood that it down-regulates the expression of EGFR， p-PI3K， and p-Akt proteins， thus further inhibits cell proliferation， and induces apoptosis.
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In this paper, tanshinone-ⅡA (Tan-IIA)/β-cyclodextrin (β-CD) inclusion complexes were prepared by saturated aqueous solution method. Based on the single factor experiment, Box-Benhnken design and response surface method were utilized to optimize the preparation procedures of tanshinone-ⅡA/β-cyclodextrin inclusion complexes. The ratio of β-CD to Tan-ⅡA, experimental temperature and time were defined as independent variables, while the yield of the inclusion complexes, encapsulation efficiency and the generalized "normalized value" were set as the response value. In addition, the inclusion complexes were characterized by infrared spectroscopy (IR) and nuclear magnetic resonance (NMR). The results showed that optimum preparation conditions for Tan-ⅡA/β-CD inclusion complex were as follows: Tan-ⅡA/β-CD ratio of 1:7, the temperature of 48 ℃ and the time of 3 h. Under the optimized conditions, the encapsulation efficiency of Tan-ⅡA/β-CD inclusion complex was 84.75%. The Tan-IIA and β-CD inclusion complex can significantly improve the dissolution of Tan-ⅡA.
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Objective To study the effects of Tan Ⅱ A on expressions of interleukin-1β(IL-1β) and interleukin-6 (IL-6) in brain tissues of neonatal rats with periventricular leukomalacia (PVL).Methods The PVL models were established by means of bilateral general cervical artery ligation followed by 0.5 h 80 mL/L oxygen and 920 mL/L nitrogen mixture exposure in 2-day rats,and then these rats were randomly divided into the model control group,the lowdose group treated with Ta Ⅱ A at 7.5 mg/(kg · d) and the high-dose group treated with Ta Ⅱ A at 15.0 mgg/(kg · d),meanwhile the false operation group was established.The rats were killed 1 hour after administration on the third day,and the brain tissues pathological changes were detected by way of HE dyeing analysis.At the same time,the expressions of IL-1β and IL-6 in brain tissues were analyzed by means of enzyme linked immunosorbent assay(ELISA).Results HE staining showed that the cells around the lateral ventricle were disarranged,as white matter appeared sparse as cribriform and ischemic changed significantly in the rats of model control group.Compared with the model control group,the ischemia of brain tissue in the low-dose group improved.Ischemia of the high-dose Tan Ⅱ A group improved more significantly than the low-dose group.ELISA results showed that the expressions of IL-1β and IL-6 in the PVL rats were significantly higher than the false operation group (all P ＜ 0.01),both of which were statistically decreased in the high-dose and the low-dose Tan Ⅱ A groups (all P ＜ 0.05) ; and there was no significant difference between the lowdose group and the high-dose group (all P ＞ 0.05).Conclusions Tan Ⅱ A has the function of improving the state of ischemia and decreasing the expressions of IL-1 β and IL-6 in tissues of PVL rats,thus it can relieve cerebral white matter lesions of neonatal rats.
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Objective To investigate the effect of CCAAT/enhancer-binding protein homologeus protein (CHOP) in Tan Ⅱ A treated acute promyelocytic leukemia (APL) cells.Methods APL cell differentiation was monitored by morphology and membrane differentiation antigens; expression of CHOP was inhibited by siCHOP; mRNA and protein expression of CHOP in Tan Ⅱ A-intervened APL cells were examined by RT-PCR and Western blot.Results Expression of CHOP was increased in Tan Ⅱ A induced differentiated APL cells (proteins levels 1.933±0.987 vs 0.537±0.110,F =114.852,P ＜ 0.01,mNRA levels 1.587±0.815 vs 0.713±0.090,F =52.256,P ＜ 0.01),combination of CHOP gene silencing with Tan Ⅱ A treatment induced strong APL cell differentiation [(50.767±1.241) ％ vs (16.167±2.122) ％,F =989.431,P ＜ 0.05] and apoptosis [(89.233±5.581) ％ vs (27.433±2.957) ％,F =308.961,P ＜ 0.05].Conclusion CHOP acts as a negative regulator in Tan Ⅱ A induced differentiation.Inhibition of CHOP may be a promising therapeutic strategy.